Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

By reconstituting lysolecithin-permeabilized hamster cells with endogenous proteins, a protein(s) which stimulated bleomycin-induced DNA repair synthesis was identified. The repair protein was inactivated by proteinase K and had an apparent molecular weight of 12 000-15 000 D. The following enzymatic activities were not detected in the partially purified DNA repair protein: general endonuclease, apurinic endonuclease, exonuclease, DNA polymerase or DNA polymerase beta-stimulating activity. The subcellular location of the DNA repair-stimulating activity was investigated by cytochalasin B enucleation; approx. 80% of the activity was associated with karyoplasts, suggesting a nuclear location. Neither the activity nor subcellular location of the repair protein fluctuated appreciably during the cell cycle, consistent with a physiological role in DNA repair. Although the function of the DNA repair protein is not yet known, this approach should be useful in identifying and characterizing mammalian DNA repair proteins.
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PMID:Identification of mammalian DNA repair factors using a reconstituted subcellular system. Partial characterization and subcellular location of a DNA repair-stimulating protein in hamster cells. 664 6

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.
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PMID:Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors. 869 23

We have established four cell lines, UW228-1, UW228-2, UW228-3 and UW443, from two posterior fossa medulloblastomas. The three UW228 sublines originated from a tumor with a diploid DNA content, while the tumor of origin of UW443 was predominantly tetraploid. Both tumors displayed areas of immunopositivity for synaptophysin and glial fibrillary acidic protein. All four cell lines have been grown as monolayers in continuous culture for 50 to 200 passages, are not contact inhibited at high density, and form colonies in soft agar. The UW228 sublines are aneuploid, have similar modal chromosome numbers, similar chromosomal duplications and identical marker chromosomes, and display loss of heterozygosity for identical sequences at the distal end of chromosome 17p. UW443 is diploid and also shows loss of heterozygosity for a distal sequence on chromosome 17p. All lines are immunopositive for two or more neurofilament proteins, three lines (UW228-1, UW228-2 and UW443) are immunopositive for synaptophysin, and none are immunopositive for glial fibrillary acidic protein. The lines differ in sensitivity to the alkylating agents 1,3-bis(2-chloroethyl)-1-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. They also differ in dependence on the DNA repair protein O6-methylguanine-DNA methyltransferase for alkylating agent resistance and in levels of the DNA repair activities apurinic/apyrimidinic endonuclease and DNA polymerase beta. These properties establish UW228-1, UW228-2, UW228-3 and UW443 as four new, phenotypically distinct medulloblastoma-derived cell lines.
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PMID:Establishment and characterization of four human medulloblastoma-derived cell lines. 886 61

DNA joining events are required for the completion of DNA replication, DNA excision repair and genetic recombination. Five DNA ligase activities, I-V, have been purified from mammalian cell extracts and three mammalian LIG genes, LIG1 LIG3 and LIG4, have been cloned. During DNA replication, the joining of Okazaki fragments by the LIG1 gene product appears to be mediated by an interaction with proliferating cell nuclear antigen (PCNA). This interaction may also occur during the completion of mismatch, nucleotide excision and base excision repair (BER). In addition, DNA ligase I participates in a second BER pathway that is carried out by a multiprotein complex in which DNA ligase I interacts directly with DNA polymerase beta. DNA ligase III alpha and DNA ligase III beta, which are generated by alternative splicing of the LIG3 gene, can be distinguished by their ability to bind to the DNA repair protein, XRCC1. The interaction between DNA ligase III alpha and XRCC1, which occurs through BRCT motifs in the C-termini of these polypeptides, implicates this isoform of DNA ligase III in the repair of DNA single-strand breaks and BER. DNA ligase II appears to be a proteolytic fragment of DNA ligase III alpha. The restricted expression of DNA ligase III beta suggests that this enzyme may function in the completion of meiotic recombination or in a postmeiosis DNA repair pathway. Complex formation between DNA ligase IV and the DNA repair protein XRCC4 involves the C-terminal region of DNA ligase IV, which contains two BRCT motifs. This interaction, which stimulates DNA joining activity, implies that DNA ligase IV functions in V(D)J recombination and non-homologous end-joining of DNA double-strand breaks. At the present time, it is not known whether DNA ligase V is derived from one of the known mammalian LIG genes or is the product of a novel gene.
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PMID:Structure and function of mammalian DNA ligases. 953 76

XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain interaction in the XRCC1-beta-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the linker-BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36-355 and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involving beta-Pol.
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PMID:Domain specific interaction in the XRCC1-DNA polymerase beta complex. 1077 72

Three mammalian genes encoding DNA ligases--LIG1, LIG3, and LIG4--have been identified. Genetic, biochemical, and cell biology studies indicate that the products of each of these genes play a unique role in mammalian DNA metabolism. Interestingly, cell lines deficient in either DNA ligase I (46BR.1G1) or DNA ligase III (EM9) are sensitive to simple alkylating agents. One interpretation of these observations is that DNA ligases I and III participate in functionally distinct base excision repair (BER) subpathways. In support of this idea, extracts from both DNA ligase-deficient cell lines are defective in catalyzing BER in vitro and both DNA ligases interact with other BER proteins. DNA ligase I interacts directly with proliferating cell nuclear antigen (PCNA) and DNA polymerase beta (Pol beta), linking this enzyme with both short-patch and long-patch BER. In somatic cells, DNA ligase III alpha forms a stable complex with the DNA repair protein Xrcc1. Although Xrcc1 has no catalytic activity, it also interacts with Pol beta and poly(ADP-ribose) polymerase (PARP), linking DNA ligase III alpha with BER and single-strand break repair, respectively. Biochemical studies suggest that the majority of short-patch base excision repair events are completed by the DNA ligase III alpha/Xrcc1 complex. Although there is compelling evidence for the participation of PARP in the repair of DNA single-strand breaks, the role of PARP in BER has not been established.
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PMID:Completion of base excision repair by mammalian DNA ligases. 1155 94

DNA polymerase beta (Polbeta) is a DNA repair protein that functions in base excision repair and meiosis. The enzyme has deoxyribose phosphate lyase and polymerase activity, but it is error prone because it bears no proofreading activity. Errors in DNA repair can lead to the accumulation of mutations and consequently to tumorigenesis. Polbeta expression has been found to be higher in tumors, and deregulation of its expression has been found to induce chromosomal instability, a hallmark of tumorigenesis, but the underlying mechanisms are unclear. In the present study, we have investigated whether ectopic expression of Polbeta influences the stability of chromosomes in a murine mammary cell line. The results demonstrate a telomere dysfunction phenotype: an increased rate of telomere loss and chromosome fusion, suggesting that ectopic expression of Polbeta leads to telomere dysfunction. In addition, Polbeta interacts with TRF2, a telomeric DNA binding protein. Colocalization of the two proteins occurs at nontelomeric sites and appears to be influenced by the change in the status of the telomeric complex.
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PMID:DNA polymerase beta interacts with TRF2 and induces telomere dysfunction in a murine mammary cell line. 1517 90

Iridovirus is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. Here, we report the complete genomic sequence of rock bream iridovirus (RBIV). The genome of RBIV was 112080 bp long and contained at least 118 putative open reading frames (ORFs), and its genome organization was similar to that of infectious spleen and kidney necrosis virus (ISKNV). Of the RBIV's 118 ORFs, 85 ORFs showed 60-99% amino acid identity to those of ISKNV. Phylogenetic analysis of major capsid protein (MCP), DNA repair protein RAD2, and DNA polymerase type-B family indicated that RBIV is closely related to red sea bream iridovirus (RSIV), Grouper sleepy disease iridovirus (GSDIV), Dwarf gourami iridovirus (DGIV), and ISKNV. The genome sequence provides useful information concerning the evolution and divergence of iridoviruses in cultured fish.
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PMID:Complete genomic DNA sequence of rock bream iridovirus. 1524 74

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT(-) phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, A. Blank, M.S. Bobola, B.A. Mueller, D.D. Kolstoe, G.A. Ojemann, M.S. Berger, Lack of the DNA repair protein O(6)-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 6941-6946]. We report here that MGMT(-) phenotype in the brain of children and adults, and the apparent increase in risk of neurocarcinogenesis, may arise during gestation. We found that MGMT activity in 71 brain specimens at 6-19 weeks post-conception was positively correlated with gestational age (P<or=0.0015). Moreover, the proportion of specimens exhibiting MGMT(-) phenotype (MGMT content<0.42 fmol/10(6)cells or 255 molecules/cell) declined progressively from 76% (16/21) at 6-8 weeks to 13% (1/8) at 15-19 weeks. All liver specimens that accompanied MGMT(-) brain (15/15) had measurable MGMT activity, demonstrating that the phenotype was not systemic in these cases. In contrast to MGMT, apurinic endonuclease, DNA polymerase beta and lactate dehydrogenase activities were found in every brain extract assayed, and showed no significant relationship with gestational age. The observed gestational pattern has at least two implications for neurocarcinogenesis. (1) Early in development, brain tissue that has MGMT(-) phenotype and is rapidly proliferating may be especially vulnerable to alkylation-induced mutations, including mutations that lead to brain tumors. (2) Persistence of prenatal MGMT deficiency into postnatal life in a sub-population of individuals may increase brain tumor risk. Our findings provide possible mechanistic insight into epidemiologic data associating maternal alkylating agent exposure with brain tumor incidence.
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PMID:O6-methylguanine-DNA methyltransferase deficiency in developing brain: implications for brain tumorigenesis. 1750 46

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle-dependent recruitment of telomere-specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S-phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase epsilon (Polepsilon) arrived at telomeres earlier than the lagging strand DNA polymerases alpha (Polalpha) and delta (Poldelta). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polepsilon, whereas S-phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polalpha. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.
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PMID:Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres. 1935 7


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